CN109486856B - Construction method and application of moso bamboo protoplast circular RNA overexpression system - Google Patents
Construction method and application of moso bamboo protoplast circular RNA overexpression system Download PDFInfo
- Publication number
- CN109486856B CN109486856B CN201811459776.4A CN201811459776A CN109486856B CN 109486856 B CN109486856 B CN 109486856B CN 201811459776 A CN201811459776 A CN 201811459776A CN 109486856 B CN109486856 B CN 109486856B
- Authority
- CN
- China
- Prior art keywords
- circular rna
- protoplast
- rna
- overexpression
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108091028075 Circular RNA Proteins 0.000 title claims abstract description 91
- 210000001938 protoplast Anatomy 0.000 title claims abstract description 69
- 230000002018 overexpression Effects 0.000 title claims abstract description 55
- 244000302661 Phyllostachys pubescens Species 0.000 title claims abstract description 36
- 235000003570 Phyllostachys pubescens Nutrition 0.000 title claims abstract description 36
- 238000010276 construction Methods 0.000 title claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 58
- 239000013612 plasmid Substances 0.000 claims abstract description 29
- 239000013598 vector Substances 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 13
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 28
- 230000009466 transformation Effects 0.000 claims description 19
- 238000006243 chemical reaction Methods 0.000 claims description 16
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 10
- 229930195725 Mannitol Natural products 0.000 claims description 10
- 239000000594 mannitol Substances 0.000 claims description 10
- 235000010355 mannitol Nutrition 0.000 claims description 10
- 229920001223 polyethylene glycol Polymers 0.000 claims description 10
- 239000002299 complementary DNA Substances 0.000 claims description 9
- 101710197633 Actin-1 Proteins 0.000 claims description 8
- 239000001110 calcium chloride Substances 0.000 claims description 8
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 8
- 238000001914 filtration Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000012154 double-distilled water Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 239000002244 precipitate Substances 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 238000012163 sequencing technique Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 5
- 102000042567 non-coding RNA Human genes 0.000 claims description 5
- 230000014509 gene expression Effects 0.000 claims description 4
- 230000001404 mediated effect Effects 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 108010059892 Cellulase Proteins 0.000 claims description 3
- 239000004677 Nylon Substances 0.000 claims description 3
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 3
- 229940106157 cellulase Drugs 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 238000009472 formulation Methods 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 238000002955 isolation Methods 0.000 claims description 3
- 108020004707 nucleic acids Proteins 0.000 claims description 3
- 102000039446 nucleic acids Human genes 0.000 claims description 3
- 150000007523 nucleic acids Chemical class 0.000 claims description 3
- 229920001778 nylon Polymers 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 238000003753 real-time PCR Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000002689 soil Substances 0.000 claims description 3
- 238000012795 verification Methods 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 230000004544 DNA amplification Effects 0.000 claims 1
- 239000008280 blood Substances 0.000 claims 1
- 210000004369 blood Anatomy 0.000 claims 1
- 238000005286 illumination Methods 0.000 claims 1
- 238000013518 transcription Methods 0.000 abstract description 9
- 230000035897 transcription Effects 0.000 abstract description 6
- 239000013604 expression vector Substances 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 108090000790 Enzymes Proteins 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 23
- 235000017166 Bambusa arundinacea Nutrition 0.000 description 11
- 235000017491 Bambusa tulda Nutrition 0.000 description 11
- 241001330002 Bambuseae Species 0.000 description 11
- 235000015334 Phyllostachys viridis Nutrition 0.000 description 11
- 239000011425 bamboo Substances 0.000 description 11
- 108090000638 Ribonuclease R Proteins 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 6
- 230000029087 digestion Effects 0.000 description 5
- 239000013642 negative control Substances 0.000 description 4
- 238000010839 reverse transcription Methods 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 108091034057 RNA (poly(A)) Proteins 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000010805 cDNA synthesis kit Methods 0.000 description 2
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 2
- 229960003669 carbenicillin Drugs 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 108091070501 miRNA Proteins 0.000 description 2
- 239000002679 microRNA Substances 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- ZYWFEOZQIUMEGL-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol;phenol Chemical compound ClC(Cl)Cl.CC(C)CCO.OC1=CC=CC=C1 ZYWFEOZQIUMEGL-UHFFFAOYSA-N 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a construction method and application of a moso bamboo protoplast circular RNA overexpression system, and belongs to the technical field of genetic engineering. The system constructs a circular RNA over-expression vector and transforms the circular RNA over-expression vector into a protoplast of the moso bamboo. The construction method comprises the following steps: amplifying a circular RNA host gene by using high-fidelity Taq enzyme, taking pUC19-35s-sGFP as a final vector, and constructing a circular RNA overexpression recombinant plasmid by using a Gateway method; a large amount of recombinant plasmids are extracted and transformed into the moso bamboo protoplast under the mediation of PEG, and the influence of the over-expression of the circular RNA on the transcription and post-transcription levels of the host genes is researched.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a construction method and application of a moso bamboo protoplast circular RNA overexpression system.
Background
Circular RNA is a special RNA molecule widely existing in organisms, is formed by gene transcription, is covalently closed at a 3 'end and a 5' end, is nonuniform in length, and generally has sequences capable of reverse complementary matching in two intron regions. Circular RNA does not have a typical poly (A) structure, and conventional transcriptome library construction generally goes through a poly (A) screening step, so that the existence of circular RNA cannot be detected, and therefore, the circular non-coding RNA is always ignored at an early stage. With the development of high-throughput sequencing technology in recent years, it has been found that circular RNA not only exists widely in organisms but also plays an important role in growth and development of the circular RNA through non-poly (A) transcriptome sequencing. The circular RNA is very stable in cells, is insensitive to exonuclease, can be combined with miRNA molecules, and can also be cut and degraded by miRNA. In addition to regulating transcription of a host gene, there is also a competitive relationship between the production of a circular RNA from a host transcript and the corresponding linear transcript. Recent studies have shown that part of the circular RNA can also enter the translational pathway.
The correlation between host genes producing circular RNA and the expression of the circular RNA is always a focus of attention, and molecular cloning technology has important significance for researching the problem, but unfortunately, for forest tree species, especially bamboo, the traditional genetic transformation takes too long time and is difficult to obtain, and the influence of the circular RNA on the transcription level of the host genes is difficult to see. The research constructs a circular RNA over-expression vector by a molecular cloning technology, converts the circular RNA into a moso bamboo protoplast under the mediation of PEG, over-expresses the circular RNA in a cell, can see the influence of the circular RNA over-expression on the transcription and post-transcription level of a host gene thereof in a short time, and greatly saves time and economic cost.
Disclosure of Invention
The invention aims to provide a construction method and application of a moso bamboo protoplast circular RNA overexpression system.
In order to achieve the purpose, the invention adopts the following technical scheme:
a construction method of a moso bamboo protoplast circular RNA overexpression system is characterized by comprising the following steps: (1) extracting moso bamboo nucleic acid; (2) verifying circular RNA; (3) constructing a circular RNA overexpression recombinant plasmid; (4) transforming the moso bamboo protoplast by the circular RNA; (5) detection of circular RNA overexpression.
The verification of the circular RNA in the step (2) comprises: designing a back-to-back primer of the circular RNA based on a circular RNA gene sequence obtained by sequencing of a transcriptome; the sequence of the circular RNA gene obtained by sequencing the transcriptome is shown as SEQ ID NO 1-SEQ ID NO 9; the sequence of the back-to-back primers of the RNA is shown in SEQ ID NO 10-SEQ ID NO 27.
The construction of the circular RNA overexpression recombinant plasmid in the step (3) comprises the following steps: amplification of a target gene; connecting a target gene to an entry vector by using a Gateway method; then the target gene on the entry vector is connected to a final vector to obtain the circular RNA over-expression recombinant plasmid.
Further, a specific primer is provided at the 5' end of the target gene during amplificationattB-A site linker; the entry vector was pDONR207 and the final vector was pUC19-35s-sGFP (Chen et al, 2011; Lin et al, 2013; Lin et al, 2014).
Furthermore, the sequence of the specific primer for amplifying the target gene is shown in SEQ ID NO: 28: 5'GGGGAC AAGTTTGTACAAAAAAGCAGGCTTCGTAAGTGGACGAAACACAAGAAGAA-3' and SEQ ID NO: 29: 5'GGGGAC CACTTTGTACAAGAAAGCTGGGTCCTTTTACCCGTCAA
TCAAACCTTA-3', wherein the underlined portion indicatesattBA recognition site.
The method for transforming the moso bamboo protoplast by the circular RNA overexpression recombinant plasmid in the step (4) comprises the following steps:
(1) isolation of protoplasts
Materials: taking the young bamboo shoots and the tender shoots which grow in the soil for 2 weeks.
Enzymolysis: longitudinally cutting the tender shoots by using a blade, putting cut strips into 20mL of enzymolysis liquid, and carrying out enzymolysis for 3h at 25 ℃ and 50rpm in a dark place; water bath at 55 deg.C for 10min, and gradually cooling to room temperature; followed by the addition of 0.2 mL of 1M CaCl20.2 mL of 10% BSA by mass/volume, and filtering the solution through a 0.45 μm filter;
and (3) filtering: after enzymolysis, washing with equal volume of W5 solution for 3-5 times, filtering with 40 μm nylon net into 50ml centrifuge tube, centrifuging at 1500rpm for 3min, standing on ice for 30min to obtain protoplast precipitate, resuspending the protoplast precipitate with MMG solution, observing with a hemacytometer under microscope, and continuously adjusting the amount of MMG solution until the protoplast concentration is 4.2 × 106Per ml;
(2) protoplast transformation
PEG-mediated protoplast transformation: in the experimental group, 10 mu g of recombinant plasmid (the volume is adjusted to 10 ul) is mixed with 100 mu L of protoplast, after the mixture is uniformly mixed, 110 mu L of freshly prepared PEG solution is immediately added for mediated transformation, and the mixture is incubated for 20min at room temperature under the dark condition; blank control group with equal amount of ddH2O replaces the recombinant plasmid, and the negative control group is added with the same amount of plasmid which does not carry the target gene.
And (3) terminating the reaction: after incubation, the reaction was stopped by slowly adding 440. mu. L W5 solution, gently inverted and mixed, centrifuged at 1,500 rpm for 3min to obtain a pellet of transformed protoplasts, and the pellet was slowly resuspended using 1.5ml W5 solution.
Culturing: finally, the transformed protoplasts were transferred to 90 mm circular petri dishes and incubated at 25 ℃ for 12-16h with light. The culture dish needs to be washed by 1ml of 1% BSA in advance, only needs to be washed, and the BSA solution in the dish is discarded after washing.
The formula of the enzymolysis liquid is as follows: 1.5% (wt/vol) Cellulase R10, 0.75% (wt/vol) Macerozyme R-10, 10 mM MES (pH 5.7), 0.6M mannitol.
W5 solution formula: 154 mM NaCl, 125 mM CaCl 25 mM KCl, 2 mM MES (pH 5.7). MMG solution formula: 0.6M mannitol, 15 mM MgCl2，4 mM MES（pH 5.7）。
MMG solution formula: 0.6M mannitol, 15 mM MgCl2，4 mM MES（pH 5.7）。
PEG solution formulation: 40% (wt/vol) PEG 4000, 0.2M mannitol, water bath 60 ℃ until the powder dissolved, gradually dropping to 40 ℃, followed by the addition of 0.1M CaCl2。
The detection of the overexpression of the circular RNA in the step (5) is as follows: extracting Total RNA of transformed Phyllostachys Pubescens protoplast, performing reverse transcription, diluting the obtained cDNA by 5 times, taking 1uL as template, taking 1uL of circular RNA back-to-back forward and reverse primers respectively, taking 25 uL of Premix Taq (TaKaRa), and adding ddH2O to 50 mu L, carrying out 40-cycle PCR reactions at 94 ℃ for 30s, 58 ℃ for 30s and 72 ℃ for 30s, detecting the over-expression of the circular RNA, and using actin-1 as an internal reference gene.
An application of a construction method of a moso bamboo protoplast circular RNA overexpression system in the influence of overexpression circular RNA on host genes.
Further, specific primers of the host genes are designed, the host genes of the protoplast circular RNA overexpression system moso bamboos are established for semi-quantitative PCR, actin-1 is used as an internal reference gene, and the influence of the overexpression circular RNA on the host genes is detected.
Furthermore, PH01000724G0700 is used as a host gene, a specific primer of the host gene is designed, and the sequence of a forward primer is SEQ ID NO. 30; the reverse primer sequence is SEQ ID NO. 31. The reaction system is as follows: mu.L of each of the circular RNA host gene-specific forward and reverse primers, 25. mu.L of Premix Taq (TaKaRa), 1uL of cDNA, plus ddH2O to 50 μ L; the reaction conditions are as follows: 40 PCR cycles at 94 ℃ for 30s, 58 ℃ for 30s, and 72 ℃ for 30 s; and detecting the influence of the overexpression of the circular RNA on the host gene.
The invention has the advantages that:
according to the annular RNA overexpression system of the moso bamboo protoplast, disclosed by the invention, the annular RNA overexpression recombinant plasmid is constructed, and the recombinant plasmid is converted into the moso bamboo protoplast, so that the rapid and stable instant conversion is realized, the influence of the annular RNA overexpression on the transcription and post-transcription regulation of the host gene can be seen in a short time, the time is greatly saved, and the economic cost is greatly saved.
Drawings
FIG. 1 shows enrichment of circular RNA after RNase R digestion, with circular RNA bands not digested with RNase R on the left, bands digested with RNase R on the right, and linear internal reference to NTB.
FIG. 2 shows the position of the bamboo seedling.
FIG. 3 shows that the young buds of Phyllostachys Pubescens are cut longitudinally and then placed in the enzymolysis solution.
FIG. 4 shows the overall conversion using blue light excitation under a fluorescence microscope and the conversion efficiency is counted.
FIG. 5 Confocal observed single protoplast transformation. "GFP fluorescence": GFP-expressing protoplasts seen under field of view using blue light excitation; "Bright field": protoplasts under a visible light field; "Merged": protoplasts with overlapping first two fields of view.
FIG. 6 overexpression of circular RNA in protoplasts. The upper part of the figure is a phyllostachys pubescens gene structure annotation (thick line is exon, thin line is intron), and the marked arrow is the position for designing the circular RNA back-to-back primers; the lower part is a gel electrophoresis picture of the cyclic RNA overexpression, in the picture, "circ-bHLH 93" is target cyclic RNA, and "actin-1" is reference gene; "shoot, protoplast, vector, Ox-circRNA" represent the bamboo shoot tissue, the blank control group, the negative control group, and the transformation group, respectively.
FIG. 7 effect of overexpression of circular RNA on host genes. The upper part of the figure is a phyllostachys pubescens gene structure annotation (thick line is exon, thin line is intron), and the marked arrow is the position for designing the host gene primer; the lower part is the semi-quantitative result of the host gene, in the figure, "line-bHLH 93" is the host gene of the circular RNA, and "actin-1" is the reference gene; "Shoot, Protoplast, Vector, Ox-circRNA" represent the bamboo Shoot tissue, the blank control group, the negative control group, and the transformation group, respectively.
Detailed Description
Example 1 extraction of Phyllostachys Pubescens nucleic acid
(1) Material treatment
Selecting bamboo shoot with 0.2cm above the ground, cutting with scalpel to obtain middle part, wrapping with tinfoil, and rapidly placing in liquid nitrogen. The material was ground using a high throughput tissue grinder (QIAGEN tissue Lyser II) and stored at-80 ℃.
(2) Extraction of bamboo Genomic DNA and Total RNA
Taking 150mg of the ground sample, and extracting Genomic DNA of bamboo shoots by using Plant Genomic DNA Kit (TIANGEN, No. DP305, China); total RNA of bamboo shoots was extracted using RNAprep Pure Plant Kit (Polysaccharides & Polyphenonics-rich) (TIANGEN, No. DP441, China). Total RNA is used for circular RNA verification, DNA is used for vector construction, and host genes are amplified.
(3) Reverse transcription of RNA
1 ug of Total RNA was reverse transcribed with Random Primer using the PrimeScript. RTM. II 1st Strand cDNA Synthesis Kit (TaKaKaKa, No. 6210A), and the resulting cDNA was diluted 5-fold and subjected to validation of circular RNA.
Example 2 validation of circular RNA
9 circular RNA sequences are obtained according to the transcriptome data, the sequences are shown as SEQ ID NO 1 to SEQ ID NO 9, and circular RNA is verified by using 'back-to-back' primers designed by PRAPI software.
Mu.g of Total RNA (adjusted to a volume of 51. mu.L) was divided equally into two tubes, one tube was subjected to RNase R digestion, and the other tube was used as a control. 3. mu.L of 10 XRNase R Reaction Buffer, 1.5. mu.L of RNase R (20U/uL) was added to the treatment tube; mu.L of 10 XRNase R Reaction Buffer, 1.5. mu.L of RNase-free water was added to the control tube. The treatment tube and the control tube were incubated in a water bath at 37 ℃ for 15min, followed by adding 30. mu.L of phenol-chloroform-isoamyl alcohol (25: 24: 1) to each tube to terminate the reaction, mixing well, and centrifuging at 13,000g for 5min at 4 ℃. Transferring the supernatant into a new 1.5mL RNase-free centrifuge tube, sequentially adding 6. mu.L of 4M LiCl, 1. mu.L of glycogen and 90. mu.L of precooled absolute ethyl alcohol (-20 ℃), uniformly mixing, standing at-80 ℃ for overnight precipitation, and centrifuging at 13,000g for 15min after precipitation to enrich RNA. Reverse transcription of RNA using Random Primer, dilution of cDNA 5 times, taking 2 uL as template, adding 15 uL Premix Taq (TaKaRa), 0.5 uL of circular RNA back-to-back forward and reverse primers, and adding ddH2And (3) performing 40-cycle PCR amplification on the total system from O to 30 mu L, detecting the size of a PCR product by using 1% agarose gel electrophoresis, and taking a reference gene NTB as a linear marker in the digestion process of RNase R. The sequences of the back-to-back primers are shown in SEQ ID NO 10-SEQ ID NO 27; the primers of the internal reference gene NTB are shown as SEQ ID NO. 32 and SEQ ID NO. 33. The results in FIG. 1 show that: after RNase R digestion, the circular RNA still exists, and the band of the internal reference gene NTB is obviously weakened after RNase R digestion.
Using cDNA of the RNase R-treated sample as a template, 5. mu.L of 10 XPCR Buffer, 5. mu.L of 2 mM dNTPs, and 3. mu.L of 25 mM MgSO4The "back-to-back" forward and reverse primers were 1.5. mu.L each, 1uL of KOD-Plus-Neo (TOYOBO, No. KOD-401), and ddH was added2O to 50. mu.L system, at 98 ℃ for 10 s, 58 ℃ for 30s, and 68 ℃ for 10 s, 35And (3) performing PCR amplification circularly, cutting gel to recover a target band, and if the sequence of the target band is consistent with the sequence of the circular RNA and is connected at the back-splicing part through sequencing, indicating that the circular RNA really exists.
EXAMPLE 3 construction of circular RNA-overexpressing recombinant plasmids
(1) Amplification of target Gene
The host gene of PH01000724G0700 (PH 01000724: 425581-426013) was selected as the target gene. Designing the 5' end of the target gene withattB-Site-specific primers. The specific primer sequences are as follows:
the forward primer is: 28, SEQ ID NO:
5’GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGTAAGTGGACGAAACACAAGAAGAA-3’
the reverse primer is: 29 in SEQ ID NO:
5’GGGGACCACTTTGTACAAGAAAGCTGGGTCCTTTTACCCGTCAATCAAACCTTA-3’
underlined and bolded partsattBA recognition site.
Using bamboo Genomic DNA as template, the above-mentioned bandattB-Positive and negative primers of site-directed linker were 2.5. mu.L each, 2 XUnique HiQTMPfu Master Mix (No Dye) (Novogene, No. NHP007L, China) 25. mu.L, plus ddH2And performing 34 PCR cycles at 98 ℃ for 10 s, 58 ℃ for 20 s and 72 ℃ for 1 min in an O to 50-mu-L system to amplify the host gene of the circular RNA. Cutting, recovering and purifying the gel to obtain the bandattB-A gene of interest of the linker sequence.
(2) Ligation of Gene of interest to entry vector
The bands were separated using BP clone (Invitrogen, No. 11789-020)attB-The target fragment of the locus is connected to a Gateway cloning universal entry vector pDONR207, the connection product is transformed into DH-5 alpha competent cells, the cells are cultured on an LB solid culture medium containing gentamicin resistance with the final concentration of 0.03 mg/mL overnight, colonies with resistance are obtained by screening, the colonies are sequenced, and downstream vector construction is carried out with correct sequence.
(3) Obtaining of circular RNA over-expression recombinant plasmid
The target gene on the entry vector is connected to a final vector pUC19-35s-sGFP (Chen et al, 2011; Lin et al, 2013; Lin et al, 2014) by using LR clonase (thermo Fisher SCIENTIFIC, No. 11791-020), the connection product is transformed into DH-5 alpha competent cells, the cells are cultured on LB solid culture medium with carbenicillin resistance at the final concentration of 0.05 mg/mL overnight, colonies with resistance are obtained by screening, and the colonies are sequenced, and the correct sequence is the recombinant plasmid which is successfully constructed finally. The recombinant Plasmid was cultured in 250 mL of LB liquid medium containing carbenicillin at a final concentration of 0.05 mg/mL for 16h, and the recombinant Plasmid was extracted in large amounts using EndoFree Maxi Plasmid Kit (TIANGEN, No. DP117, China) so that the recombinant Plasmid concentration was 1 mg/mL or more.
Example 4 circular RNA protoplast transformation
(1) Isolation of protoplasts
Materials: the young bamboo shoots growing in the soil for 2 weeks were picked up and the tender shoots were observed as shown in FIG. 2.
Enzymolysis: the tender shoots were cut longitudinally with a blade, and the cut pieces were placed in an enzymatic hydrolysate for enzymatic hydrolysis at 25 ℃ and 50rpm for 3 hours in the dark as shown in fig. 3. The formula of the enzymolysis liquid is as follows: 1.5% (wt/vol) Cellulase R10, 0.75% (wt/vol) Macerozyme R-10, 10 mM MES (pH 5.7), 0.6M mannitol, water bath at 55 ℃ for 10min, gradually cooled to room temperature. Followed by the addition of 10 mM CaCl20.1% (wt/vol) BSA, filtered using a 0.45 μm filter.
And (3) filtering: after enzymolysis, washing with equal volume of W5 solution for 5 times, filtering with 40 μm nylon net, standing and centrifuging at 1500rpm for 3min, standing on ice for 30min to obtain protoplast precipitate, resuspending the protoplast with MMG solution, observing with a blood count plate under microscope, and adjusting the protoplast concentration to 4.2 × 10 by adding MMG solution6One per ml. W5 solution formula: 154 mM NaCl, 125 mM CaCl 25 mM KCl, 2 mM MES (pH 5.7). MMG solution formula: 0.6M mannitol, 15 mM MgCl2，4 mM MES（pH 5.7）。
(2) Protoplast transformation
PEG-mediated protoplast transformation: experimental group 10. mu.g of recombinant plasmid (volume adjustment)To 10 uL) and 100 uL protoplast, then immediately adding 110 uL of freshly prepared PEG solution to mediate the transformation after mixing uniformly, and incubating for 20min at room temperature under the dark condition; blank control group with equal amount of ddH2O replaces the recombinant plasmid, and the negative control group is added with the same amount of plasmid which does not carry the target gene. PEG solution formulation: 40% (wt/vol) PEG 4000, 0.6M mannitol, water bath 60 ℃ until the powder dissolved, gradually dropping to 40 ℃, followed by the addition of 0.1M CaCl2。
And (3) terminating the reaction: after incubation, the reaction was stopped by slowly adding 440. mu. L W5 solution, gently mixing by inversion, and after centrifugation at 1,500 rpm for 3min, the transformed protoplast pellet was obtained and slowly resuspended using 1.5mL of W5 solution.
Culturing: finally, the transformed protoplasts were transferred to a petri dish and incubated at 25 ℃ for 16h with light. The dishes were washed beforehand with 1mL of 1wt% BSA, and after washing, the BSA solution in the dishes was discarded.
(3) Method for efficiently counting protoplast transformation efficiency
Sucking 10 mu L of the transformed protoplast on a glass slide, observing by using a Zeiss fluorescent microscope at a 10-fold or 20-fold lens, and counting the transformation efficiency; transformation efficiency = (number of GFP-fluorescent protoplasts in field/total number of protoplasts) × 100% as shown in fig. 4, and at least three fields were counted to calculate the average transformation efficiency. The single protoplast transformation was then observed using Confocal, as shown in FIG. 5.
Example 5 detection of circular RNA overexpression
1) Extracting the Total RNA of the transformed moso bamboo protoplast: total RNA was extracted using the RNAprep Pure Plant Kit (Polysaccharides & Polyphenonics-rich) (TIANGEN).
2) Reverse transcription: the RNA extracted in step 1) was reverse-transcribed into cDNA using PrimeScript. sup.II 1st Strand cDNA Synthesis Kit (TaKaKa), diluted 5-fold for use.
3) Detection of circular RNA overexpression: taking cDNA 1uL diluted by 5 times obtained in the step 2) as a template, taking 1uL of each of 'back-to-back' forward and reverse primers, 25 uL of Premix Taq (TaKaRa), and adding ddH2O to 50. mu.L, as per 9440 cycles of PCR reactions at 30s, 58 ℃ 30s, and 72 ℃ 30s were performed to detect the overexpression of circular RNA, as shown in FIG. 6. The actin-1 gene is internal reference, and the primers of the internal reference gene actin-1 are shown as SEQ ID NO. 34 and SEQ ID NO. 35.
Example 6 detection of the Effect of overexpression of circular RNA on host genes
Taking PH01000724G0700 as a host gene, designing a specific primer of the host gene, wherein a forward primer sequence: 30 of SEQ ID NO: PH01000724G 0700-F5'-CGTCGTCAGCTGCTTCAAC-3'; reverse primer sequence SEQ ID NO: 31: PH01000724G0700-R: 5'-TCTACAAGCAGCCTCCTCCT-3'; semi-quantitative PCR of the host gene was performed. mu.L of each forward and reverse primer of the circular RNA host gene, 25. mu.L of Premix Taq (TaKaRa), and 1uL and ddH of cDNA2O to 50. mu.L, and 40 cycles of PCR at 94 ℃ for 30s, 58 ℃ for 30s, and 72 ℃ for 30s, it was found that overexpression of the circular RNA decreased the expression level of the host gene, as shown in FIG. 7.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
Construction method and application of <120> Phyllostachys pubescens protoplast circular RNA overexpression system
<130> 35
<160> 35
<170> PatentIn version 3.3
<210> 1
<211> 253
<212> DNA
<213> PH01002436G0250
<400> 1
gagctcgtcc ggatggagtg cgagcggtgg gcgcacaagg gggtgaacat cacgtaccag 60
atccgggaga accgcaaggg gtacaaggcc ggcgcgctca aggagggcat gaagcacggg 120
tacgtgcgcg agtgcgagta cgtggccatc ttcgacgccg acttccagcc ggaccccgac 180
ttcctccgcc gcaccatccc gttcctcatg aacaacccgg agatcgccct cgtccaggcg 240
cgctggaggt tcg 253
<210> 2
<211> 264
<212> DNA
<213> PH01002004G0190
<400> 2
agagagtacg aggagttcaa ggtgcggatc aacgcgctgg tcgcgaaggc gcagaaggtg 60
cccgccgagg gctggatcat gaaggacggt acgccgtggc ccggcaacaa cacccgcgac 120
caccccggca tgatccaggt gttcttggga cacagcggag gccacgacac cgagggcaac 180
gagctgcccc gcctggtgta cgtctcccgt gagaagcggc ccgggttcca gcaccacaag 240
aaggccggcg ccatgaacgc tctc 264
<210> 3
<211> 429
<212> DNA
<213> PH01000391G0210
<400> 3
gataacaaag atgcaaggca tgttaagaca tatgaagttg cattgaagga caaagacttt 60
gttgagggtc cttggtcgca gaacaattta gacaatggtg ctggtctgtt aatacctgta 120
ccggctccac ttggtggtgt cataattatt ggcgaggaga caatagttta ctgcaatgct 180
aatgctacat ttaaagccat accaatcaaa caatccatcg taagagctta cggacgggtt 240
gacccagatg gttctcgtta tttactcggt gataatacgg gcactttgca tttacttgtc 300
cttacccacg aacgagaaag ggtaactggt ttgaaaattg agtacttggg agagacttca 360
gttgcatcat caatatcata tcttgataat ggtgttgtct atgtcggttc tcgatttggt 420
gattcacag 429
<210> 4
<211> 581
<212> DNA
<213> PH01003949G0150
<400> 4
agcatggggc tactatccca tcattatctt ctcacggcag cacacctcag tactccacat 60
atggtggata ccagggtaca agcaaaaaaa ttgaaatccc aaatggaagg gttggtgtca 120
tcattggaaa atctggggac actataaaat atctccaaca gcagtcggga gctaagattc 180
aagtaacaag agacatggat gctgaacctg gctcacagac aagaccggtt gagctttcag 240
gcactcctga ccagataagc aaagctgagc agatgatcaa tgctgttctg gcagatgctg 300
atgctgcttc atctggcact gtctctaccc ggaaatacaa tgcatctcaa ccaggtgctg 360
agcaattcca aatgaaaatt gccaacaaca aggtgggtct ggttattgga aagggtggtg 420
agactataaa aaacatgcaa atcaaatctg gagcccatat acaggtgaga cctttgcatt 480
tgcctcctgg tgatacttca actgaaagaa cagtgtatat tgaaggcaca caagatcaaa 540
ttgaaatcgc aaagcagcta gtgaatgagg ttaccagtga g 581
<210> 5
<211> 392
<212> DNA
<213> PH01000798G0710
<400> 5
ggaagccgcc gctggactgg cacacgcgca agcggatcgc cgtcggcacg gcgcggggcc 60
tgctgtacct gcacgagcag tgcgacccca agatcatcca ccgcgacgtc aaggccgcca 120
acgtgctgct cgacgagtgc cacgaggccg tcgtcggaga cttcggcctc gccaagctgc 180
tcgaccacgg cgactcgcac gtcaccacgg cggtgcgcgg caccgtgggc cacatcgcgc 240
cggagtacct ctccacaggg cagtcgtcgg agaagaccga cgtgttcggg ttcgggatcc 300
tgctgctcga gctcgtcacg ggacagcgcg cgctcgaggt cggcaaggcc tcggggatca 360
gccagagcca gaagggggtc atgcttgatt gg 392
<210> 6
<211> 392
<212> DNA
<213> PH01000048G0850
<400> 6
ggaagccgcc gctggactgg cacacgcgca agcggatcgc cgtcggcacg gcgcggggcc 60
tgctgtacct gcacgagcag tgcgacccca agatcatcca ccgcgacgtc aaggccgcca 120
acgtgctgct cgacgagtgc cacgaggccg tcgtcggaga cttcggcctc gccaagctgc 180
tcgaccacgg cgactcgcac gtcaccacgg cggtgcgcgg caccgtgggc cacatcgcgc 240
cggagtacct ctccacaggg cagtcgtcgg agaagaccga cgtgttcggg ttcgggatcc 300
tgctgctcga gctcgtcacg ggacagcgcg cgctcgaggt cggcaaggcc tcggggatca 360
gccagagcca gaagggggtc atgcttgatt gg 392
<210> 7
<211> 863
<212> DNA
<213> PH01002058G0150
<400> 7
gcacatatta ccgcaggcaa gagaaggctt ctgagcccag cagcagtgta cttacacctg 60
gagttgcaac ttgttccggc tgccttgcac ttcctgttgc acttaggatt tgtggttgtg 120
gatacagcag tgctatcaag gactgggaaa cttttcgttt ctgcaagcaa acatcaaaaa 180
aggagagaac agttataata attcttgatt cagatgatga ggatggaaac actgcaggaa 240
gtaaacagtt gtcaactgtg aagaacgaac aactgacaac atctgaattg gctggagaca 300
aaaacactca aattgttcct tacggtcaaa gtgcggattc aacgaatcag cttcctttgc 360
aaaccagctt gcaaccatct attgagtttg agagaattat gttgcagaaa agacctgatg 420
aggagcgtat tcaagatcta gtggctgaga ggaaacgacg gaaatcagac cacagctccc 480
aggtgaatgg agacgctgca accgccccaa gacaaagaaa aagaaaaggc gagcagaatc 540
cagcagcagt tgatttcccc ttcggtttac aacaaactga tagcccctca gaaacctaca 600
agcctatgga gaaagaggag aaaccaggaa aaacaagtga tcatcttgag gatctttgga 660
atggttttac actggctatg gaaagctcaa agcttgacac atctgaagag gtagccaatg 720
ataaacaagt ggacaatgaa aaagaggtgg acaatgactg caatcatgac attcgtgttc 780
acgaagacct gggccatgta tgtcgtgtct gtggtatgat tgtgagaagt gctgattcaa 840
taattgatta tcagtggaaa aag 863
<210> 8
<211> 540
<212> DNA
<213> PH01001683G0200
<400> 8
gaaggtgatc cccgcctgtc aagttttgga ctaatgaaga acagccgtga cggaaaaagt 60
tatagcacca acctggctta caccccacca gagtttctac gaaccggcag agtcatcccc 120
gagagtgtga tatatagcta cggaacagtt ctgttggatc ttttgagcgg gaagcacatt 180
cctcctagtc acgcacttga tttgataaga ggaaagaata tactgttgct gatggattcc 240
tccttagaag ggcaatatgc caatgaagac gcttcaaaat tagttgatct tgcatcgaaa 300
tgtttgcagt ttgaagctag ggacagaccc aatataaagt atctcttgtc ttccgttggg 360
cctcttcaga agcaaaagga ggtggcatca catgtgttaa tgggcattac aaaagccacg 420
tcagtgttgc caaccattct ttcgccactt gggaaggcct gtgccagtat ggaccttaca 480
gcggtacatg atatattgct taaaacgggt tacaaagatg atgaaggtgc cgaaaacgag 540
<210> 9
<211> 621
<212> DNA
<213> PH01000724G0700
<400> 9
gtccattggg atgaaccttc ttccatcttg cgtccagaga gagtatcacc atgggatttg 60
gaacctcttg ttgcaactac tccttcgaac tcccaaccta tgcagaggaa caagcggcca 120
cggccatctg tcttgccctc accaacagcc aatctttctg cacttggtat gtggaaacct 180
tcggttgagt cttcagcttt ctcatatggt gaatcacaac gtggacgaga cccttatcca 240
tcacccaatt tctctaccac tgcaaaggcc aactctctta gcttctgtgg caatagtcaa 300
gtgaccagtg tttcgccgaa ttcaatgtat cggcctaacc aagtggaaag tgtcacagat 360
tcttttgctc cagttgtaaa caaagatttg ggagaaagga gacagggtac tgggattggc 420
tacagacttt tcgggattca acttattgac aatttcaatg cagaaggaac ttcaccagtg 480
gttactgtgt ctggaacagt gggcaatgat cgcccggttg tgtctttaga ggctgagtct 540
gatcagcatt ctgagcctga gaaatcatgt ctgagatctc atcaggagtt gcaaagtagg 600
caaatcagga gctgcacaaa g 621
<210> 10
<211> 20
<212> DNA
<213> PH01002436G0250 -F
<400> 10
caccatcccg ttcctcatga 20
<210> 11
<211> 20
<212> DNA
<213> PH01002436G0250 -R
<400> 11
cgaagatggc cacgtactcg 20
<210> 12
<211> 18
<212> DNA
<213> PH01002004G0190 -F
<400> 12
ccccgcctgg tgtacgtc 18
<210> 13
<211> 18
<212> DNA
<213> PH01002004G0190 -R
<400> 13
gtggtcgcgg gtgttgtt 18
<210> 14
<211> 20
<212> DNA
<213> PH01000391G0210 -F
<400> 14
tgtccttacc cacgaacgag 20
<210> 15
<211> 20
<212> DNA
<213> PH01000391G0210 -R
<400> 15
cgaccaagga ccctcaacaa 20
<210> 16
<211> 20
<212> DNA
<213> PH01003949G0150 -F
<400> 16
tgcatttgcc tcctggtgat 20
<210> 17
<211> 20
<212> DNA
<213> PH01003949G0150 -R
<400> 17
agctcaaccg gtcttgtctg 20
<210> 18
<211> 20
<212> DNA
<213> PH01000798G0710 -F
<400> 18
ggagaagacc gacgtgttcg 20
<210> 19
<211> 20
<212> DNA
<213> PH01000798G0710 -R
<400> 19
atgatcttgg ggtcgcactg 20
<210> 20
<211> 20
<212> DNA
<213> PH01000048G0850 -F
<400> 20
aataccaatt cacccgctgg 20
<210> 21
<211> 22
<212> DNA
<213> PH01000048G0850 -R
<400> 21
gtggaataca cctcgaggat ca 22
<210> 22
<211> 20
<212> DNA
<213> PH01002058G0150 -F
<400> 22
agaggtggac aatgactgca 20
<210> 23
<211> 20
<212> DNA
<213> PH01002058G0150 -R
<400> 23
gtgcaacagg aagtgcaagg 20
<210> 24
<211> 20
<212> DNA
<213> PH01001683G0200 -F
<400> 24
cctcctagtc acgcacttga 20
<210> 25
<211> 20
<212> DNA
<213> PH01001683G0200 -R
<400> 25
aactctggtg gggtgtaagc 20
<210> 26
<211> 20
<212> DNA
<213> PH01000724G0700 -F
<400> 26
acccgggtgg agatctactg 20
<210> 27
<211> 20
<212> DNA
<213> PH01000724G0700 -R
<400> 27
cgatgtcctg tccatctcgg 20
<210> 28
<211> 56
<212> DNA
<213> Artificial sequence
<400> 28
ggggacaagt ttgtacaaaa aagcaggctt cgtaagtgga cgaaacacaa gaagaa 56
<210> 29
<211> 54
<212> DNA
<213> Artificial sequence
<400> 29
ggggaccact ttgtacaaga aagctgggtc cttttacccg tcaatcaaac ctta 54
<210> 30
<211> 19
<212> DNA
<213> Artificial sequence
<400> 30
cgtcgtcagc tgcttcaac 19
<210> 31
<211> 20
<212> DNA
<213> Artificial sequence
<400> 31
tctacaagca gcctcctcct 20
<210> 32
<211> 23
<212> DNA
<213> NTB-F
<400> 32
aatagctgtc cctggaggag ttt 23
<210> 33
<211> 23
<212> DNA
<213> NTB-R
<400> 33
tcttgtttga caccgaagag gag 23
<210> 34
<211> 23
<212> DNA
<213> actin-1-F
<400> 34
atacgcttcc tcacgctatt ctt 23
<210> 35
<211> 23
<212> DNA
<213> actin-1-R
<400> 35
ccgagcttct cctttatgtc cct 23
Claims (7)
1. A construction method of a moso bamboo protoplast circular RNA overexpression system is characterized by comprising the following steps: (1) extracting moso bamboo nucleic acid; (2) verifying circular RNA; (3) constructing a circular RNA overexpression recombinant plasmid; (4) converting the Phyllostachys pubescens protoplast by the circular RNA overexpression recombinant plasmid; (5) detecting the overexpression of the circular RNA;
the verification of the circular RNA in the step (2) comprises the following steps: designing a back-to-back primer of the circular RNA based on a circular RNA gene sequence obtained by sequencing of a transcriptome; the sequence of the circular RNA gene obtained by sequencing the transcriptome is shown as SEQ ID NO. 9; the sequences of the circular RNA back-to-back primers are shown as SEQ ID NO. 26 and SEQ ID NO. 27.
2. The method for constructing the annular RNA overexpression system of the moso bamboo protoplast according to claim 1, wherein the method comprises the following steps: the construction of the circular RNA overexpression recombinant plasmid comprises the following steps: amplification of a target gene; connecting a target gene to an entry vector by using a Gateway method; then the target gene on the entry vector is connected to a final vector to obtain the circular RNA over-expression recombinant plasmid.
3. The method for constructing the annular RNA overexpression system of the moso bamboo protoplast according to claim 2, wherein the expression system comprises: the 5' end of the specific primer is provided withattB-A site linker; the entry vector is pDONR 207; the final vector is pUC19-35s-sGFP。
4. The method for constructing the annular RNA overexpression system of the moso bamboo protoplast according to claim 3, wherein the expression system comprises: the specific primer sequence during the target gene amplification is shown as SEQ ID NO: 28: 5'GGGGACAAGTTTGTACAAAAAA GCAGGCTTCGTAAGTGGACGAAACACAAGAAGAA-3' and SEQ ID NO: 29: 5'GGGGACCACTTTGTACAAGAAA GCTGGGTCCTTTTACCCGTCAATCAAACCTTA-3', wherein the underlined portion indicatesattBA recognition site.
5. The method for constructing the phyllostachys pubescens protoplast circular RNA overexpression system according to claim 1, wherein the transformation of the phyllostachys pubescens protoplast by the circular RNA overexpression recombinant plasmid comprises the following steps:
(1) isolation of protoplasts
Materials: taking moso bamboo seedlings and tender shoot parts which grow for 2 weeks in soil;
enzymolysis: longitudinally cutting the tender shoots by using a blade, putting cut strips into 20mL of enzymolysis liquid, and carrying out enzymolysis for 3h at 25 ℃ and 50rpm in a dark place; water bath at 55 deg.C for 10min, and gradually cooling to room temperature; followed by the addition of 0.2 mL of 1M CaCl20.2 mL of 10% BSA by mass/volume, and filtering the solution through a 0.45 μm filter;
and (3) filtering: after enzymolysis, washing with equal-volume W5 solution for 3-5 times, filtering with 40 μm nylon net into 50ml centrifuge tube, centrifuging at 1500rpm for 3min, standing on ice for 30min, removing supernatant with pipette to obtain protoplast precipitate, resuspending the protoplast precipitate with MMG solution, observing with a blood counting chamber under microscope, and continuously adjusting the amount of MMG solution until the protoplast concentration is 4.2 × 106Per ml;
(2) protoplast transformation
PEG-mediated protoplast transformation: mixing 10 mu g of recombinant plasmid with 100 mu L of protoplast, mixing uniformly, immediately adding 110 mu L of freshly prepared PEG solution to mediate conversion, and incubating for 20min at room temperature under a dark condition; blank control group with equal amount of ddH2O instead of the recombinant plasmid, negativeAdding an equal amount of plasmid not carrying the target gene into the control group;
and (3) terminating the reaction: after incubation, slowly adding 440 mu L W5 solution to terminate the reaction, gently inverting and mixing evenly, centrifuging at 1500rpm for 3min to obtain transformed protoplast precipitate, and slowly resuspending the precipitate by using 1.5ml W5 solution;
culturing: finally, transferring the transformed protoplast to a culture dish, and incubating for 12-16h at 25 ℃ under illumination; the culture dish needs to be washed by 1mL of 1wt% BSA solution in advance, and the BSA solution in the dish is discarded after washing;
the formula of the enzymolysis liquid is as follows: cellulase R10 with the mass volume percentage concentration of 1.5 percent, Macerozyme R-10 with the mass volume percentage concentration of 0.75 percent, 10 mM MES, 0.6M mannitol and pH of 5.7;
w5 solution formula: 154 mM NaCl, 125 mM CaCl2，5 mM KCl ，2 mM MES，pH 5.7；
MMG solution formula: 0.6M mannitol, 15 mM MgCl2，4 mM MES，pH 5.7；
PEG solution formulation: PEG 4000 with a mass volume percentage concentration of 40%, 0.2M mannitol, water bath at 60 ℃ until the powder is dissolved, gradually reducing the temperature to 40 ℃, and then adding 0.1M CaCl2。
6. The method for constructing the annular RNA overexpression system of the moso bamboo protoplast according to claim 1, wherein the detection of the annular RNA overexpression in the step (5) is as follows: extracting Total RNA of the transformed Phyllostachys Pubescens protoplast, reverse transcribing the Total RNA into cDNA, diluting by 5 times, taking 1uL as a template, taking 1uL of circular RNA back-to-back forward and reverse primers respectively, obtaining 25 uL of Premix Taq, and adding ddH2O to 50 mu L, carrying out 40-cycle PCR reactions at 94 ℃ for 30s, 58 ℃ for 30s and 72 ℃ for 30s, detecting the over-expression of circular RNA, and taking actin-1 gene as an internal reference.
7. The application of the method for constructing the annular RNA overexpression system of the moso bamboo protoplast according to claim 1 in the influence of the overexpression of the annular RNA on host genes, which is characterized in that: designing a specific primer of a circular RNA host gene, carrying out semi-quantitative PCR on the host gene of moso bamboo of which a protoplast circular RNA overexpression system is established, and detecting the influence of the overexpression circular RNA on the host gene by using actin-1 as an internal reference gene; the host base is a gene with a sequence shown as SEQ ID NO. 9, a specific primer of a circular RNA host gene, and a forward primer sequence is shown as SEQ ID NO. 30; the reverse primer sequence is shown as SEQ ID NO. 31.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811459776.4A CN109486856B (en) | 2018-11-30 | 2018-11-30 | Construction method and application of moso bamboo protoplast circular RNA overexpression system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811459776.4A CN109486856B (en) | 2018-11-30 | 2018-11-30 | Construction method and application of moso bamboo protoplast circular RNA overexpression system |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109486856A CN109486856A (en) | 2019-03-19 |
| CN109486856B true CN109486856B (en) | 2021-11-02 |
Family
ID=65698143
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811459776.4A Expired - Fee Related CN109486856B (en) | 2018-11-30 | 2018-11-30 | Construction method and application of moso bamboo protoplast circular RNA overexpression system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109486856B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108546670A (en) * | 2018-04-19 | 2018-09-18 | 贵州大学 | A kind of method for transformation of the preparation method of Sorghum Protoplast and the protoplast of preparation |
| CN111849858B (en) * | 2020-07-20 | 2023-01-10 | 浙江农林大学 | Preparation of moso bamboo protoplast and establishment of transient transformation system |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105145355A (en) * | 2015-09-08 | 2015-12-16 | 中国林业科学研究院亚热带林业研究所 | Phyllostachys edulis protoplast culture method |
| CN107025385A (en) * | 2016-11-07 | 2017-08-08 | 龚畅 | A kind of design method of circular rna primer |
-
2018
- 2018-11-30 CN CN201811459776.4A patent/CN109486856B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105145355A (en) * | 2015-09-08 | 2015-12-16 | 中国林业科学研究院亚热带林业研究所 | Phyllostachys edulis protoplast culture method |
| CN107025385A (en) * | 2016-11-07 | 2017-08-08 | 龚畅 | A kind of design method of circular rna primer |
Non-Patent Citations (3)
| Title |
|---|
| PEG介导番木瓜叶肉原生质体瞬时表达体系的建立;霍鹏等;《分子植物育种》;20150428(第04期);106-113 * |
| Transient expression for functional gene analysis using Populus protoplasts;Biyue Tan et al;《Plant Cell Tiss Organ Cult》;20130201;11-18 * |
| Widespread noncoding circular RNAs in plants;Chu-Yu Ye et al;《New Phytologist》;20151231;88-95 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109486856A (en) | 2019-03-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113005139B (en) | Application of transcription factor PsMYB1 in regulation and control of synthesis of peony petal anthocyanin | |
| CN109486856B (en) | Construction method and application of moso bamboo protoplast circular RNA overexpression system | |
| CN106754957B (en) | OsSCAMP13 gene, encoding protein, application of stress resistance and acquisition method | |
| CN113265406A (en) | Soybean FDL12 gene editing site and application thereof | |
| CN112522279A (en) | Coding sequence of rice grain type gene OsGL8 gene and application | |
| CN114807138B (en) | Plant annular RNA over-expression vector, construction method and application thereof | |
| CN113929759B (en) | Upstream regulatory factor IbERF73 and application thereof in regulation and control of IbWD40 expression of purple sweet potato | |
| CN115925848A (en) | Dendrobium ERF transcription factor gene DoERF5 and application thereof | |
| CN112430260B (en) | Application of abundant protein DoLEA36 in late embryonic development or coding gene thereof in regulation and control of plant callus formation | |
| CN111621508B (en) | Tobacco terpene synthase NtTPS7 gene and vector and application thereof | |
| CN113774080B (en) | Method for creating high-anthocyanidin horticultural ornamental poplar by using miR156 | |
| CN109971744B (en) | Malan blue BcTSA gene and encoded protein and application thereof | |
| CN112501185A (en) | Application of pineapple transcription factor AcWRKY28 in salt resistance of plants | |
| CN107723293A (en) | A kind of cotton fiber development related gene GbWRKY32 and its expression vector and application | |
| CN108841853B (en) | Carrot genetic transformation method using glyphosate as screening agent | |
| CN113234753A (en) | Cultivation, identification and application of maize microfilament depolymerizing factor ADF7 transgenic plant | |
| CN113215187A (en) | Method for rapidly obtaining fragrant rice material by using CRISPR/Cas9 technology | |
| CN104988176B (en) | Method for improving gum content of eucommia ulmoides | |
| CN116751791B (en) | Application of PvPSK3 gene in improving genetic transformation efficiency of gramineous plants | |
| CN117210490B (en) | PCHR gene for regulating and controlling malus plant self-flower fructification and application thereof | |
| KR102551064B1 (en) | Novel U6 promoter separated form grapevine and use of the same | |
| CN114891802B (en) | Application of OsDUF6 gene and encoding protein thereof in rice salt tolerance breeding | |
| CN112852805B (en) | Preparation method of rice miRNA homozygous lethal mutant | |
| WO2015196874A1 (en) | Cotton plant event ac20-3 and primer and method for the detection thereof | |
| CN109251926B (en) | Promoter SPP specifically expressed in plant stem and/or leaf stalk, expression vector and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20211102 |